This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Intracellular Ca2+-release channels are essential for mobilizing Ca2+ from intracellular stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR) into the cytoplasm of cells. A wide variety of cellular processes, including muscle contraction, cell proliferation, secretion, fertilization, and cell differentiation, rely on transient changes in cytoplasmic Ca2+ concentration [1]. This proposal focuses in inositol 1,4,5-trisphosphate receptors (IP3Rs), homotetrameric Ca2+-release channels occurring in the ER/SR membranes of virtually all cells. IP3 is the primary IP3R ligand, which synergizes with Ca2+ to promote IP3 channel opening. Type-1 IP3R (IP3R1), the predominant isoform in the ER of cerebellar Purkinje cells, is the focus of this project. IP3R1 forms homotetramers of ~1.3 MDa in the cerebellar ER [2]. Structural studies of IP3Rs have been hampered by both its size and its interaction with membrane lipids in its native conformation. X-ray crystallography and NMR spectroscopy are poorly suited methodologies to study massive integral membrane proteins due to this double complexity. In this project, we aim to determine 3D structure of IP3R1 channel at subnanometer resolution and to delineate structural determinants of its gating. Such structural information is ultimately needed for the development of pharmacological strategies to interfere with the channel protein in disease states. In our structural studies we exploit single particle electron cryomicroscopy of the detergent-solubilized purified channel protein and computer reconstruction techniques.